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SubmityourarticletothisJournal(http://www.hh.um.es/Instructions.htm)Analysis of wt1a reporter line expression levels during proepicardium formation in

the zebrafish

Authors: Laura Andrés-Delgado, María Galardi-Castilla, Nadia Mercader and LuisSantamaría DOI:10.14670/HH-18-238Articletype:JOURNALARTICLEAccepted:2020-07-07Epubaheadofprint:2020-07-07

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Analysis of wt1a reporter line expression levels during proepicardium formation in the zebrafish

Laura Andrés-Delgado1,2,#, María Galardi-Castilla2, Nadia Mercader2,3 and Luis

Santamaría1

1 Department of Anatomy, Histology and Neuroscience. School of Medicine. Autonoma

University of Madrid. 28029 Madrid. Spain.

2. Development of the epicardium and its role during regeneration laboratory, Nacional

Center of Cardiovascular Research Carlos III. 28029 Madrid, Spain.

3 Institute of Anatomy, University of Bern, 3000 Bern 9, Switzerland.

# Corresponding author: Laura Andrés-Delgado, PhD. Departamento de Anatomía,

Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid. C/

Arzobispo Morcillo, 4, 28029 Madrid, España. e-mail: laura.andresd@uam.es

Short running title: Wt1a levels in pericardial tissues

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Key Words: Zebrafish, pericardial tissues, Cavalieri´s volume estimation, Bmp signaling Summary: The epicardium is the outer mesothelial layer of the heart. It covers the

myocardium and plays important roles in both heart development and regeneration. It is

derived from the proepicardium (PE), groups of cells that emerges at early developmental

stages from the dorsal pericardial layer (DP) close to the atrio-ventricular canal and the venous

pole of the heart-tube. In zebrafish, PE cells extrude apically into the pericardial cavity as a

consequence of DP tissue constriction, a process that is dependent on Bmp pathway signaling.

Expression of the transcription factor Wilms tumor-1, Wt1, which is a leader of important

morphogenetic events such as apoptosis regulation or epithelial-mesenchymal cell transition,

is also necessary during PE formation. In this study, we used the zebrafish model to compare

intensity level of the wt1a reporter line epi:GFP in PE and its original tissue, the DP. We found

that GFP is present at higher intensity level in the PE tissue, and differentially wt1 expression

at pericardial tissues could be involved in the PE formation process. Our results reveal that

bmp2b overexpression leads to enhanced GFP level both in DP and in PE tissues.

Introduction The heart develops from the lateral plate mesoderm, and is one of the first organs to

form and acquire its function. The primitive heart tube is composed of two layers, the

myocardium and the endocardium, and during embryogenesis the third cellular layer of the

heart, which is the epicardium, is developed. The epicardium covers the myocardium and

influences it through paracrine signals that promote its growth and function (Olivey and

Svensson, 2010; Perez-Pomares and de la Pompa, 2011). Also, epicardial-derived cells

(EPDCs) can differentiate into other cell types as cardiac fibroblasts or adipocytes (Chau et

al., 2014) and can contribute to the formation of the valves (Wessels et al., 2012; Duenas et

al., 2017; Simoes and Riley, 2018). After heart injury, EPDCs can be involved in several

aspects of tissue repair and regeneration (Kennedy-Lydon and Rosenthal, 2015).

The epicardium, also known as the visceral pericardium, has an extracardiac origin. It

derives from the proepicardium (PE), transient groups of cells that emerge from the dorsal

parietal pericardium (DP) in locations close to the atrio-ventricular canal (avc) and the venous

pole (vp) of the heart tube. It appears around the time of heart looping and after the onset of

heart beating (Schulte et al., 2007; Maya-Ramos et al., 2013; Kennedy-Lydon and Rosenthal,

2015). After PE is formed, the PE cells translocate and attach to the myocardial surface to form

the epicardial layer (Manner et al., 2001).

How the PE is formed and how PE cells reach heart tube differs between models. In

chicken, a temporary bridge forms between the PE and the myocardium and PE cells migrate

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to the myocardial surface (Nahirney et al., 2003). Near aortic sac pericardium also contributes

to epicardium formation in chicks (Perez-Pomares et al., 2003). In mice, a dual model was

proposed. PE cells are transferred to the myocardium through direct contacts between the PE

and the myocardium, and PE cells can be released into the pericardial cavity as floating

clusters, which adhere to the myocardium (Rodgers et al., 2008; Li et al., 2017).

In zebrafish, three different PE populations were described (Peralta et al., 2013; Peralta

et al., 2014): avcPE cluster; vpPE cluster; and arterial pole epicardial precursor cells (apPE),

which are single pericardial cells at arterial pole site that directly contact the myocardium but

barely contribute to epicardium. Once the avcPE and the vpPE cell clusters form, pericardial

fluid flow generated by heartbeat stimulates PE cell aggregates to detach and reach the heart

tube, spreading atop the myocardium to form a new tissue layer: the epicardium (Peralta et al.,

2013). In combination with the floating PE clusters, a PE cellular bridge attached to the heart

tube was also described in zebrafish (Plavicki et al., 2014). The differences obtained by the

groups might be due to the different techniques and markers used. Plavicki et al. used

fluorescent reporter driven by regulatory sequences of tcf21, a pan-epicardial marker, while

the regulatory element of wt1a (epi:GFP reporter line), which labels 70% of the PE cells, was

used by Peralta and colleagues. Thereby, the combination of the results indicate that in

zebrafish also a dual mechanism model for epicardial formation could be happening. PE

formation in zebrafish is regulated by Bone morphogenetic protein (Bmp) pathway (Liu and

Stainier, 2010), and Bmp signaling leads PE clusters formation by promoting actin cytoskeleton

dynamics (Andres-Delgado et al., 2019).

There is a continuous rearrangement of tissue layers in the heart that affects cardiac

morphogenesis (Andres-Delgado and Mercader, 2016; Collins and Stainier, 2016; Steed et al.,

2016; Ocana et al., 2017). In zebrafish, the PE forms by a mechanism that involves the

constriction of the DP layer and the apical extrusion of PE cell clusters to the pericardial cavity

(Andres-Delgado et al., 2019). This suggests that an epithelial-mesenchymal-like transition

(EMT) process could be taking place in the DP cells that transform into PE (Serluca, 2008; Wu

et al., 2010; Tandon et al., 2016; Andres-Delgado et al., 2019). Wilms tumor 1 (WT1) has been

implicated in regulating the EMT cell fate equilibrium in multiple contexts, including heart

development (Smart et al., 2011) or cancer (Chau and Hastie, 2012). PE and epicardial layer

presents expression of the transcription factor wt1 in the larval zebrafish, and its presence is

required for the determination of PE specification (Serluca, 2008). Wt1 expression in the PE

has been postulated to be crucial for the development of this structure (Moore et al., 1999;

Pombal et al., 2008; Serluca, 2008; Zeng et al., 2011). In mammals and avian epicardium,

WT1 has been related with the maintenance of an intact epithelial morphology (Martinez-

Estrada et al., 2010; Bax et al., 2011), and mutant analysis revealed an essential function of

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WT1 during epicardial development as the null mice are not viable (Kreidberg et al., 1993;

Moore et al., 1999; Carmona et al., 2001).

Here, we found that in the wt1a reporter line epi:GFP, GFP level is significantly higher in

PE compared to the DP layer. Bmp signaling affects positively to the GFP intensity level in the

different zebrafish pericardial tissues and favors the expression of wt1a.

Objectives -To determine whether in zebrafish, tissue volume and GFP intensity level of the wt1a

reporter line epi:GFP are similar in the dorsal pericardium compared to the proepicardium.

-To identify whether the overexpression of bmp2b regulates tissue volume and GFP

intensity level of the wt1a reporter line epi:GFP in the dorsal pericardium and in the

proepicardium of developing zebrafish.

-To determine whether wt1a expression level in dorsal pericardium and in the

proepicardium is affected by Bmp2b.

Materials and methods Zebrafish strains and husbandry

All experiments were approved by the Community of Madrid “Direccion General de

Medio Ambiente”, Spain. Animals were housed and experiments were performed in

accordance with Spanish bioethical regulations for the use of laboratory animals. Zebrafish

(Danio rerio) were maintained at a water temperature of 28 ºC. The following fish were used:

wild-type AB strain (ZIRC, Eugene, OR, USA); Et(-26.5Hsa.WT1-gata2:EGFP)cn1 (hereafter

termed epi:GFP) in which GFP expression is controlled by the regulatory elements of wilms

tumor 1a (wt1a), and recapitulates its expression pattern (Peralta et al., 2013) and

Tg(hsp70l:bmp2b)fr13 (Chocron et al., 2007).

Heat shock

In order to increase bmp2b levels at a specific developmental stage, the transgenic line

Tg(hsp70l:bmp2b)fr13, which overexpressed bmp2b under a heat-shock promoter, were heat

shocked as follows: heat shocks were performed to the embryos at 39ºC in preheated water

for 1 h at 26, 35 and 48 hpf (Andres-Delgado et al., 2019).

Animals treated with heat shocks were genotyped after confocal image acquisition. This

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allowed unbiased comparison and blinded quantification of experimental and control groups.

Immunofluorescence

60 hpf embryos were fixed overnight in 4% paraformaldehyde in PBS, washed in 0.1%

PBS Tween20 (Sigma-Aldrich) and permeabilized with 0.5% Triton-X100 (Sigma-Aldrich) in

PBS for 20 min. Several washing steps were followed by 2 h blocking with 5% goat serum, 5%

BSA, 20 mM MgCl2 in PBS followed by overnight incubation with the primary antibody at 4ºC.

Secondary antibodies were diluted 1:500 in PBS and incubated for 3 h. Nuclei were

counterstained with DAPI (Invitrogen) for 30 min. After several washes, embryos were

mounted in Vectashield (Vector).

The antibodies for immunofluorescence detection were as follows: anti-myosin heavy

chain (MF20, 2147781 DSHB) at a 1:20 dilution, anti-GFP (1010 Aveslab) at 1:1000, anti-β-

catenin (610153, BD Transduction Laboratories) at 1:200 and anti-pH3 (06-570, Merck) at

1:100. Secondary antibodies were the following: anti-mouse IgG2b 568 (Invitrogen) and anti-

chicken 488 (Life Technologies).

Embryos were imaged with a Zeiss 780 confocal microscope fitted with a 20× objective

1.0 NA with a water immersion lens. Z-stack images of the whole pericardial cavity were taken

every 5 μm. Maximum projections of Z-stack images were 3D reconstructed in whole-mount

views using Imaris software (Bitplane Scientific Software). The ventral pericardial layer was

digitally removed to provide a clearer view of the heart tube and DP. Optical sections of 1 z-

slice were also shown in some figures.

Definition of the dorsal pericardial tissue and proepicardium

PE clusters have been described to emerge from the DP layer, appearing from a region

close to the avc in the vp of the heart tube surrounding area (Peralta et al., 2013). Because

there is no specific marker that allows differentiating PE from pericardial cells, position and

shape has been used to define PE. The dorsal pericardial tissue has a flat morphology and

was labeled as the actual DP, while groups of cells with a rounded morphology on the DP close

to the avc region were referred as the PE (Peralta et al., 2013; Andres-Delgado et al., 2019).

To estimate the roundness index (R index) of DP and avcPE, the height (h) and width

(w) of those tissues were measured on epi:GFP 60 hpf zebrafish. In pericardial cavity images

(2D optical sections from an images-stack), the h and the w of the right DP area and of the

avcPE was measured using Image J software. A minimum of 4 images per animal were

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measured. The ratio h / w gives the R index. The closer the R index is to 1, closer to the

circumference will be the shape of the structure.

Digital isolation of the dorsal pericardium

In epi:GFP embryos, all EPDCs express GFP (Peralta et al., 2014). To favor the

visualization of DP tissue in the 3D reconstructions of pericardial cavities, the ventral

pericardium was removed from all images of the stack. Imaris software (Bitplane Scientific

Software) Surface-Function was used to create a mask of the heart without the ventral

pericardium, and 3D reconstruction of the heart cavity with the DP digitally isolated was done.

Volume estimation

Calculation of structures volume was performed using stereological Cavalieri’s estimator

(Gundersen et al., 1988; Cruz-Orive, 1999) from Image J: Volumest PlugIn Merzin, Markko

("Applying stereological method in radiology. Volume measurement. "Bachelor's thesis.

University of Tartu. 2008). Images from a stack which comprises all the slices were the region

of interest (DP or avcPE) appears were used. The volume was estimated by the formula:

𝑽 = 𝒕× 𝑨𝒏

𝟏

t = actual thickness of slice. t = 5 µm.

n = number of slices where the region of interest appears.

A = area of interest were calculated using the Cavalieri´s estimator and were expressed

in µm2.

Intensity quantification

Anti-GFP-488 immunofluorescence intensity quantification (epi:GFP line) was calculated

for DP and PE regions. A minimum of 8 slices from an images-stack were quantified, per

animal, using Image J: Integrated Optical Density. The product of Area by Mean Gray Value

(average gray value within the selected region) determined the GFP-Wt1a intensity level in the

DP tissue or avcPE areas per animal.

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In situ hybridization

In situ hybridization on whole-mount 60 hpf zebrafish embryos was performed as

described (Jowett and Lettice, 1994), using riboprobes against full coding sequence of wt1a

and GFP cDNAs (Peralta et al., 2013). Larvae were fixed in 4% PFA overnight, dehydrated in

methanol series and stored at -20°C until use. On day 1, embryos were bleached in 1,5% of

H2O2 in methanol, rehydrated, and digested with proteinase-K 10 µg.ml-1 for 17 min.

Endogenous alkaline phosphatase was blocked with triethanolamine 0,1 M, pH 8 with 0.25%

of acetic anhydride for 20 min and re-fixed in 4% PFA for 20 min. After washing, they were

pre-hybridized at 68°C for at least 1 h. The antisense riboprobe was added at 0.5 µg.ml-1. After

hybridization, several washes were done before overnight incubation with 1:4000 dilution of

anti digoxigenin-AP antibody (Roche, 11093274910) in blocking solution (Roche,

11096176001). Embryos were developed in BM-Purple until signal was detected.

Statistical analysis

Student’s t test for comparisons between two groups was used. The level of significance

selected was P < 0.05. Calculations were made with Microsoft Excel and GraphPad. P-values

are indicated either in the figure legends, the main text or summarized.

Results Measurement of dorsal pericardium and proepicardium volume

At 60 hpf, the main PE cluster in zebrafish, the avcPE, appears on the DP tissue near

the atrio-ventricular area (Fig. 1a). Z-stack images were taken every 5 µm, and 3D maximum

projection reconstruction of the pericardial cavity was shown (Fig. 1a-1). 3D heart was rotated

45º on X-axis (Fig. 1a-2) to allow a better view of the PE at the avc site. Ventral pericardium

was digitally removed (Fig. 1a-3).

The roundness of the pericardial tissues was determined using the Roundness Index

(R). R is 0.028 ± 0.02 for DP tissue, whereas for PE is 12.5-fold more: 0.35 ± 0.083. PE tissue

is more globular shaped than the flat DP tissue (Fig. 1b,c).

Continuing with the characterization of the PE tissue, we determined that despite the

roundness of PE, the DP layer volume was significantly larger (133000 ± 67000 µm3) than in

the avcPE cluster (10700 ± 2070 µm3, n = 6 animals) (Fig. 2a,b).

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Proepicardial tissue presents increased GFP intensity level

Intensity level of GFP, as a reflection of the Wt1a level, using the epi:GFP zebrafish line,

was measured both in DP and in PE tissue. Our results determined that PE GFP intensity per

volume was significantly higher than in the DP layer of the same animal (n = 6 animals) (Fig.3).

Bmp2b overexpression increases proepicardium volume

In control epi:GFP animals subjected to heat shock (HS), the PE was normally formed

and visible at 60 hpf. Overexpression of bmp2b using the transgenic line Tg(hsp70l:bmp2b)

crossed into epi:GFP, resulted in a 2-fold enlargement in the PE tissue volume at 60 hpf (Fig.

4a). PE tissue volume in control was 10700 ± 2070 µm3 compared with a volume of 23000 ±

2450 µm3 upon bmp2b overexpression (n= 6 animals; P-value < 0.0001) (Fig. 4b). However,

bmp2b overexpression does not affect the DP tissue layer volume.

We next aimed to assess whether the enhancement in the PE volume is due to PE cell

proliferation favored by Bmp2b. To do this, we evaluated the expression of phospho-histone 3

(pH3), a biomarker that labels cells in mitosis. We detected that bmp2b overexpression does

not affect the number of proliferating cells, neither in the DP (Fig. 5a,b) nor in the PE (Fig.

5a,c).

bmp2b overexpression increases GFP intensity level in the pericardium

We next wanted to investigate whether the Bmp pathway signaling can affect the

intensity level of GFP, as a reflection of the Wt1a level, in the pericardial tissues. Using 60 hpf

epi:GFP animals overexpressing bmp2b, GFP intensity per volume was higher in the PE

cluster than in the DP tissue of the same bmp2b-overexpressed animal (n = 6 animals) (Fig.

6a,b).

We explored whether the enlargement of avcPE cluster volume observed upon bmp2b

overexpression occurs concomitantly with an increase in GFP level. GFP intensity, was

significantly different in DP compared to PE, both in control and upon bmp2b overexpression

(Fig. 7a,b). Besides, we showed that Bmp2b can increase the GFP intensity level in the PE

tissue (18.1 ± 4.01 Arbitrary Units (AU) in control versus 47.8 ± 14.7 AU in the PE of bmp2b

overexpressing embryos, P-value = 0.0007)) (Fig. 7b). Interestingly, Bmp2b was also able to

enhance GFP intensity level in the DP tissue (1.96 ± 0.796 AU in control versus 10.3 ± 5.3 AU

in bmp2b-overexpressed DP, P-value = 0.0025) (Fig. 7b).

Finally, we compared the GFP intensity level per tissue volume. In PE, this value was

much higher than in their DP layer (Fig. 7a,c). In the DP tissue, this signal was higher upon

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bmp2b overexpression: 0.023 ± 0.008 AU/µm3 in control compared to 0.075 ± 0.029 AU/µm3

in bmp2b overexpress DP, P-value= 0.0021 (Fig. 7c). Regarding the PE, GFP level also was

higher in animals overexpressing bmp2b (Fig. 7b), but taking into account that the volume of

PE cluster is double sized by Bmp2b, significance here was not reported (0.647 ± 0.358

AU/µm3 in control animals versus 0.968 ± 0.362 AU/µm3 in the PE of embryos overexpressing

bmp2b) (Fig. 7c).

bmp2b overexpression favors wt1a expression level in the pericardium

To confirm the observations in the epi:GFP transgenic line, we performed whole-mount

in situ hybridization against GFP and the native wt1a, both in control and upon bmp2b

overexpression (Fig. 8a). GFP and wt1a mRNA were detected at 60 hpf in zebrafish PE cluster

and to a lesser extent in the DP tissue (Fig. 8b,c). Bmp2b increased the expression of GFP in

both the PE and in the DP (Fig. 8b). wt1a expression is also affected by Bmp2b (Fig. 8c).

Discussion

Here, we show that bmp2b overexpression enhances wt1a level in the pericardial

tissues, and Wt1a could be involved in the PE volume increasing observed in animals

overexpressing bmp2b.

To our knowledge, there is no specific marker for PE cells or other pericardial-derived

tissues. Epicardium, DP and PE are all positive for the same pericardial lineage markers (Zeng

et al., 2011; Peralta et al., 2014; Meilhac et al., 2015). The lack of markers makes difficult the

differentiation of PE from DP cells, the position and rounder shape of the PE clusters was used

to discriminate them from DP cells (Peralta et al., 2013; Plavicki et al., 2014; Andres-Delgado

et al., 2019). In zebrafish epicardial cells are heterogeneous based on the expression of

candidate genes (Cao et al., 2016; Weinberger et al., 2020), but specific markers are required

for further studies in the field. Wt1 expression in the PE has also been described as crucial for

its development (Moore et al., 1999; Pombal et al., 2008; Serluca, 2008; Zeng et al., 2011),

and in zebrafish wt1 transcription factor is expressed in PE and epicardium, and is necessary

for the PE formation (Serluca, 2008). Using zebrafish epi:GFP line that recapitulates wt1a

expression (Peralta et al., 2013), we show that the GFP intensity signal and the wt1a

expression level are significantly higher in avcPE cluster compared to DP tissue, which can be

used to differentiate PE cells from DP tissue and will help to describe new PE characteristics.

In mice, WT1 presence in the PE organ is mediated by NX2.5 (Zhou et al., 2008), and in chicks,

proepicardial markers such as Tbx18 or Wt1 upregulation are mediated by FGF8 and Snail1

via the Twist1 molecule (Schlueter and Brand, 2009; Braitsch and Yutzey, 2013). Forced

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expression of TWIST1 leads to PE formation (Schlueter and Brand, 2013) while absence of

Tbx18 in embryos also exhibits proepicardial abnormalities (Kao et al., 2015). Wt1 has

important functions during PE formation across species, and its expression level in the

pericardial tissues might influence PE emergence.

Changes in the tissue specification mechanisms have to take place in the DP cells to

allow its differentiation into PE (Andres-Delgado et al., 2019). Our results could indicate that

higher expression of Wt1 in the PE benefits its EMT-like process of roundness from the flat DP

layer. WT1 is key in the morphogenesis of different structures (Chau and Hastie, 2012), and

has been implicated in the specification of EMT phenotype (Huang et al., 2012; Artibani et al.,

2017). During development of certain mesodermal tissues, Wt1 regulates mesenchymal cell

maintenance, and depending on the context, loss of Wt1 can induce mesenchymal to epithelial

cell shift or the reverse process (Ocana et al., 2012; Rivas et al., 2013). In epicardium, WT1

acts in maintaining an intact epithelial cell morphology (Martinez-Estrada et al., 2010; Bax et

al., 2011), while WT1 null mice are not viable (Kreidberg et al., 1993; Moore et al., 1999;

Carmona et al., 2001). In human epicardial cells, WT1 knockdown stimulates the loss of

epithelial morphology, the reduction of the expression of E-cadherin, α4-integrin and VCAM-1

and the significantly increase of Snai1 (Bax et al., 2011), but the opposite results were also

described (Martinez-Estrada et al., 2010; von Gise et al., 2011). Together, all these findings

suggest that WT1 plays a major role regulating the balance between epithelial-mesenquimal

phenotypes, two fundamental cell states in mesodermal tissues.

Apical extrusion is a cellular mechanism that controls epithelial layer homeostasis, and

it takes place in the intestine while preserving its barrier function (Norden et al., 2009;

Simovitch et al., 2010; Ritchie et al., 2012) or during embryogenesis to control cell number.

Thereby, supernumerary cells have to be eliminated towards the apical side, with the

consequent death of extruded cells due to loss of survival factors (Eisenhoffer et al., 2012;

Marinari et al., 2012). On the contrary, in cancer, transformed cells can live and extrude. WT1

overexpression has also been implicated in cancer progression due to its anti-apoptotic effect

(Hartkamp et al., 2010). In PE formation process, the increased Wt1 level might be maintaining

the PE cell in an alive state until attachment to myocardium.

Bmp pathway has been found to be involved in PE formation process in mice (del Monte

et al., 2011) and chicken (Schlueter et al., 2006) where after Bmp2 addition Wt1 is

downregulated in PE. In zebrafish, mutants for the Bmp receptor acvr1l or animals inhibited

with a Bmp antagonist do not form a PE, whereas bmp2b overexpression extends PE (Liu and

Stainier, 2010; Andres-Delgado et al., 2019). Here, we show than in zebrafish Bmp2b

increased wt1a in the PE and interestingly, in the DP tissue as well. bmp2b overexpression

also correlates with a bigger PE, suggesting that increased Wt1 level in DP and PE affects

positively to the PE formation process in zebrafish.

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In conclusion, we propose a model in which Bmp pathway signaling promotes Wt1 level

in the DP tissue, which enhances the process of PE extrusion. Maintenance of high level of

Wt1 in the PE cells might favors cells mesenchymal state and apoptosis inhibition until

attachment to myocardium.

Acknowledgements We are grateful to the Animal Facility and Microscopy Unit at Nacional Center of

Cardiovascular Research Carlos III.

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Figure Legends: Fig. 1. Proepicardium is more rounded than the dorsal pericardial tissue. (a) 3D

maximum projections of a 60 hpf zebrafish heart. epi:GFP embryos immunostained for GFP

(green) and myosin heavy chain (MHC) (red). a1, Distance between slices is 5 µm; a2, 3D

image a1 was rotated 45º in the X-axis; a3, in image a2 ventral pericardium had been digitally

removed to provide a clearer view of the DP layer and the PE. Arrowheads, DP layer or avcPE

cluster. (b) Top panel, optical section of a 60 hpf zebrafish heart, and zoomed images below.

epi:GFP embryos immunostained for GFP (green), β-catenin (red) and nuclei were

counterstained with DAPI (blue). Arrowheads, DP layer or avcPE cluster. White lines illustrate

the height and width of DP layer or avcPE. (c) Quantification of DP and PE tissue roundness

index (R) (n=6 animals). at, atrium; avc, atrio-ventricular canal; DP, dorsal pericardium; h,

height; hpf, hours post fertilization; PE, proepicardium; R, roundness index; v, ventricle; w,

width; Scale bar: 50 µm. Data are mean ± s.d., unpaired two-tailed Student's t-test. ***P <

0.001.

Fig. 2. Dorsal pericardial tissue volume is bigger than in proepicardium. (a) Top

panel, optical section of a 60 hpf zebrafish heart, and zoomed images below. epi:GFP embryos

immunostained for GFP (green), myosin heavy chain (MHC) (red) and nuclei were

counterstained with DAPI (blue). Arrowheads, DP layer or avcPE cluster. White ellipses

indicate the position of DP tissue or avcPE. (b) Quantification of DP and PE volume (n=6

animals). at, atrium; DP, dorsal pericardium; hpf, hours post fertilization; PE, proepicardium; v,

ventricle; Scale bar: 50 µm. Data are mean ± s.d., unpaired two-tailed Student's t-test. ***P <

0.001.

Fig. 3. Proepicardium presents increased GFP intensity level compared to the

dorsal pericardial tissue. Quantification of DP and PE GFP intensity signal per tissue volume

in 6 60 hpf animals. AU, arbitrary units; DP, dorsal pericardium; PE, proepicardium; Data are

mean ± s.d., unpaired two-tailed Student's t-test. ***P < 0.001.

Fig. 4. bmp2b overexpression increases proepicardium volume. (a) 3D maximum

projections of 60 hpf zebrafish hearts. epi:GFP embryos immunostained for GFP (green) and

myosin heavy chain (MHC) (red). Control compared with an animal overexpressing bmp2b.

Arrowheads, DP layer or avcPE cluster. (b) Quantification of DP and PE volume from

conditions shown in a (n=6 animals). at, atrium; DP, dorsal pericardium; hpf, hours post

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fertilization; PE, proepicardium; v, ventricle; Scale bar: 50 µm. Data are mean ± s.d., unpaired

two-tailed Student's t-test. ***P < 0.001, ns-non significant.

Fig. 5. (a) bmp2b overexpression does not affect pericardial cell proliferation. (a)

epi:GFP embryos immunostained for GFP (green), myosin heavy chain (MHC) (red), pH3

(white) and nuclei counterstained with DAPI (blue). Top panels, 3D projection of 60 hpf

zebrafish hearts, middle panels optical sections and zoomed images below. Control compared

with an animal overexpressing bmp2b. Arrowheads, DP layer or avcPE cluster. Arrows, DP

pH3+ cells. Yellow arrowheads, PE cells, all negative for pH3. (b, c) Quantification of pH3+

cells number in the DP (b) or in the PE (c) (n=6 animals). at, atrium; hpf; hours post fertilization;

PE, proepicardium; v, ventricle. Scale bars: 50 µm (20 µm in zoomed a). Data are mean ±

s.d.., unpaired two-tailed Student's t-test. ns-non significant.

Fig. 6. Upon bmp2b overexpression proepicardium presents increased GFP

intensity level compared to the dorsal pericardial layer. (a) Top panel, optical section of a

60 hpf bmp2b overexpressing zebrafish heart, and zoomed images below. epi:GFP embryos

immunostained for GFP (green), myosin heavy chain (MHC) (red) and nuclei were

counterstained with DAPI (blue). Arrowheads, DP layer or avcPE cluster. White ellipses

indicate the position of DP tissue or avcPE. (b) Quantification of DP and PE GFP intensity

signal per tissue volume in 6 60 hpf animals. at, atrium; AU, arbitrary units; DP, dorsal

pericardium; hpf, hours post fertilization; PE, proepicardium; v, ventricle; Scale bar: 50 µm.

Data are mean ± s.d., unpaired two-tailed Student's t-test. ***P < 0.001.

Fig. 7. bmp2b overexpression increases GFP intensity level both in the

proepicardium and in the dorsal pericardial tissue. (a) Top panel, optical sections of 60 hpf

zebrafish hearts, and zoomed images below. epi:GFP embryos immunostained for GFP

(green), myosin heavy chain (MHC) (red) and nuclei were counterstained with DAPI (blue).

Control compared with an animal overexpressing bmp2b. Arrowheads, DP layer or avcPE

cluster. (b) Quantification of DP and PE GFP intensity. (c) Quantification of DP and PE GFP

intensity per tissue volume (n=6 animals). at, atrium; AU, arbitrary units; DP, dorsal

pericardium; hpf, hours post fertilization; PE, proepicardium; v, ventricle; Scale bar: 50 µm.

Data are mean ± s.d., unpaired two-tailed Student's t-test. **P < 0.01, ***P < 0.001, ns-non

significant.

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Fig. 8. bmp2b overexpression affects wt1a expression level in the pericardial tissues. (a) Whole-mount in situ hybridization for GFP and wt1a in control and bmp2b

overexpressing animals at 60 hpf. White arrows, DP. Black arrows, avcPE. A representative

animal is shown for each condition. Number of animal with the expression phenotype shown

are indicated in the right corner. (b) Quantification of DP and PE GFP in situ hybridization

intensity mean (n=8 animals in control and 7 bmp2b overexpressing animals). (c) Quantification of DP and PE wt1a in situ hybridization intensity mean (n=5 animals in control

and 9 bmp2b overexpressing animals). AU, arbitrary units; DP, dorsal pericardium; hpf, hours

post fertilization; PE, proepicardium; Scale bar: 50 µm. Data are mean ± s.d., unpaired two-

tailed Student's t-test. *P < 0.05, **P < 0.01, ns-non significant.

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